SUMMARY Clostridium difficile infection (CDI) is now the country's leading cause of antibiotic-associated diarrhea and has increased acute care facility costs by $4.8 billion per year. CDI was originally restricted to health care settings, but is now responsible for community-acquired infections as well. The infection toll is now approaching 500,000 cases per year, leading to >14,000 fatalities. It has clearly become urgent to find novel ways of preventing and treating infection. In fact, the US Department of Health and Human Services has set a goal of reducing hospitalization due to C. difficile infection (CDI) by 30% by 2020. In most cases, the onset of CDI is due to treatment of a patient with a broad-spectrum antibiotic, a treatment that undercuts the protective effect of the normal microbial flora. Unfortunately, the standard treatment for CDI also depends on antibiotics, all of which leave a significant number of patients uncured. Moreover, 20-30% of patients who appear to have been cured suffer one or more cases of recurrent infection. The recurrence is undoubtedly due to the fact that the antibiotics used to treat CDI do not allow the microbiota to re-establish itself in a timely and efficient manner. Novel, non-antibiotic, anti-infective approaches are clearly needed to reduce the impact of CDI. The form of C. difficile that initiates infection is the spore. Since C. difficile virulence factors are produced by vegetative cells, germination of the spore and its return to vegetative growth is the first step in pathogenesis. Importantly, spore germination depends on certain human bile acids and is inhibited by others. In healthy people, the microbiota eliminates the most active pro-germination bile acids and produces the bile acids most effective in blocking germination and vegetative cell growth. We have synthesized analogs of both activating and inhibitory bile acids and identified lead compounds that block spore germination in vitro >400-times more efficiently than does the most inhibitory natural bile acid. We propose here to synthesize a further set of compounds that are designed to maintain germination inhibition while reducing the likelihood of metabolism, absorption and toxicity. We will test the ability of these compounds to prevent germination both in vitro and in vivo, and to resist metabolism by the gut flora. The new lead compounds will then be tested for their basic pharmacokinetics and potential toxicity in mice and for their ability to inhibit primary and recurrent infection. The new lead compounds created in this nine-month project will subsequently be tested in animals for more detailed pharmacokinetics as a means of preparing them for IND approval and a Phase I clinical trial.